1. Field of the Invention
The present invention relates generally to the field of transmembrane receptors, more particularly to seven segment transmembrane G protein-coupled receptors, and most particularly to the serotonin (5-HT) receptors. Through genetic mutational techniques, the amino acid sequences of the native 5-HT.sub.2A and 5-HT.sub.2C receptors have been modified so that the receptors exist in a constitutively activated state exhibiting both a greater response to agonists and a coupling to the G Protein second messenger system even in the absence of agonist. A method for constitutively activating G protein-coupled 5-HT receptors in general is also disclosed.
2. Description of Related Art
The research interest in G protein-coupled cell surface receptors has exploded in recent years as it has been apparent that variants of these receptors play a significant role in the etiology of many severe human diseases. These receptors serve a diverse array of signalling pathways in a wide variety of cells and tissue types. Indeed, over the past 20 years, G protein-coupled receptors have proven to be excellent therapeutic targets with the development of several hundred drugs directed towards activating or deactivating them.
G protein-coupled receptors form a superfamily of receptors which are related both in their structure and their function. Structurally the receptors are large macromolecular proteins embedded in and spanning the cell membrane of the receiving cell and are distinguished by a common structural motif. All the receptors have seven domains of between 22 to 24 hydrophobic amino acids forming seven .alpha. helixes arranged in a bundle which span the cell membrane substantially perpendicular to the cell membrane. The transmembrane helixes are joined by chains of hydrophilic amino acids. The amino terminal and three connecting chains extend into the extracellular environment while the carboxy terminal and three connecting chains extend into the intracellular environment. Signalling molecules are believed to be recognized by the parts of the receptor which span the membrane or lie on or above the extracellular surface of the cell membrane. The third intracellular loop joining helixes five and six is thought to be the most crucial domain involved in receptor/G protein coupling and responsible for the receptor selectivity for specific types of G proteins.
Functionally, all the receptors transmit the signal of an externally bound signalling molecule across the cell membrane to activate a heterotrimeric transducing protein which binds GDP (guanosine diphosphate). Upon activation, the bound GDP is converted to GTP (guanosine triphosphate). The activated G protein complex then triggers further intracellular biochemical activity. Different G proteins mediate different intracellular activities through various second messenger systems including, for example, 3'5'-cyclic AMP (cAMP), 3'5'-cyclic GMP (cGMP), 1,2-diacylglycerol, inositol 1,4,5-triphosphate, and Ca.sup.2+. Within the human genome, several hundred G protein-coupled receptors have been identified and endogenous ligands are known for approximately 100 of the group. While the seven transmembrane motif is common among the known receptors, the amino acid sequences vary considerably, with the most conserved regions consisting of the transmembrane helixes.
Binding of a signalling molecule to a G protein-coupled receptor is believed to alter the conformation of the receptor, and it is this conformational change which is thought responsible for the activation of the G protein. Accordingly, G protein-coupled receptors are thought to exist in the cell membrane in equilibrium between two states or conformations: an "inactive" state and an "active" state. In the "inactive" state (conformation) the receptor is unable to link to the intracellular transduction pathway and no biological response is produced. In the altered conformation, or "active" state, the receptor is able to link to the intracellular pathway to produce a biological response. Signalling molecules specific to the receptor are believed to produce a biological response by stabilizing the receptor in the active state.
Discoveries over the past several years have shown that G protein-coupled receptors can also be stabilized in the active conformation by means other than binding with the appropriate signal molecule. Four principal methods have been identified: 1) molecular alterations in the amino acid sequence at specific sites; 2) stimulation with anti-peptide antibodies; 3) over-expression in in vitro systems; and 4) over-expression of the coupling G proteins. These other means simulate the stabilizing effect of the signalling molecule to keep the receptor in the active, coupled, state. Such stabilization in the active state is termed "constitutive receptor activation".
Several features distinguish the constitutively activated receptors. First, they have an affinity for the native signalling molecule and related agonists which is typically greater than that of the native receptors. Second, where several known agonists of varying activity (to the native receptor) were known, it was found that the greater the initial activity of the agonist, the greater was the increase in its affinity for the constitutively activated receptor. Third, the affinity of the constitutively activated receptor for antagonists is not increased over the affinity for the antagonist of the native receptor. Fourth, the constitutively activated receptors remain coupled to the second messenger pathway and produce a biological response even in the absence of the signalling molecule or other agonist.
The importance of constitutively activated receptors to biological research and drug discovery cannot be overstated. First, these receptors provide an opportunity to study the structure of the active state and provide insights into how the receptor is controlled and the steps in receptor activation. Second, the constitutively active receptors allow study of the mechanisms by which coupling to G proteins is achieved as well as how G protein specificity is determined. Third, mutated constitutively active receptors are now recognized in disease states. Study of constitutively activated receptors has demonstrated that many mutations may lead to constitutive activation and that a whole range of activation is possible. Fourth, the existence of constitutively active receptors provides a novel screening mechanism with which compounds which act to increase or decrease receptor activity can be identified and evaluated. Such compounds may become lead compounds for drug research. Finally, studying the affect of classical antagonists (compounds previously identified as, in the absence of agonist, binding to the receptor but causing no change in receptor activitiy, and, in the presence of agonist, competitively decreasing the activity of a receptor) and other drugs used as treatments on the constitutively active receptors has led to the discovery that there are compounds, inverse agonists, which decrease the constitutive activity of the active state of the receptors but which have no or little affect on the inactive state. The difference between antagonists, which act on the inactive state, and inverse agonists, which act on the active state, is only discernable when the receptor exhibits constitutive activity. These inverse agonists, identifiable with constitutively active receptors, present an entirely new class of potential compounds for drug discovery.
About 10 years ago, it was recognized that neurotransmitter receptors can be divided into two general classes depending on the rapidity of their response. Fast receptors were identified with ion channels and mediate millisecond responses while slower receptors were identified with G protein-coupled receptors. These G protein-coupled receptors include certain subtypes of the adrenergic as well as the muscarinic cholinergic (M1-M5), dopaminergic (D1-D5), serotonergic (5-HT1, 5-HT2, 5-HT4-5-HT7) and opiate (.delta., .kappa., and .mu.) receptors. Each of these G protein-coupled neurotransmitter receptors has been associated with profound changes in mental activity and functioning, and it is believed that abnormal activity of these receptors may contribute to certain psychiatric disorders. Consequently, the elucidation of the mechanism of action of these receptors has been the focus of vigorous research efforts.
Serotonin receptors are of particular importance. Serotonin-containing cell bodies are found at highest density in the raphe regions of the pons and upper brain stem. However, these cells project into almost all brain regions and the spinal column. Serotonin does not cross the blood-brain barrier and is synthesized directly in neurons from L-tryptophan. In the CNS serotonin is thought to be involved in learning and memory, sleep, thermoregulation, motor activity, pain, sexual and aggressive behaviors, appetite, neuroendocrine regulation, and biological rhythms. Serotonin has also been linked to pathophysiological conditions such as anxiety, depression, obsessive-compulsive disorders, schizophrenia, suicide, autism, migraine, emesis, alcoholism and neurodegenerative disorders. Presently several drugs are used to modify serotonin receptors: 1) 5-HT1: sumatriptan for treatment of migraine, ipsapirone and buspirone for treatment of anxiety; 2) 5-HT2: clozapine and risperidone for treatment of schizophrenia; and 3) 5-HT3: odanestron for the prevention of emesis in chemotherapy.
To date, fourteen serotonin receptors have been identified in 7 subfamilies based on structural homology, second messenger system activation, and drug affinity for certain ligands. The 5-HT.sub.2 subfamily is divided into 3 classes: 5-HT.sub.2A, 5-HT.sub.2B, and 5-HT.sub.2C. 5-HT.sub.2A and 5-HT.sub.2C receptor antagonists are thought to be useful in treating depression, anxiety, psychosis, and eating disorders. 5-HT.sub.2A and 5-HT.sub.2C receptors exhibit 51% amino acid homology overall and approximately 80% homology in the transmembrane domains. The 5-HT.sub.2C receptor was cloned in 1987 and led to the cloning of the 5-HT.sub.2A receptor in 1990. Studies of the 5-HT.sub.2A receptor in recombinant mammalian cell lines revealed that the receptor possessed two affinity states, high and low. Both the 5-HT.sub.2A and 5-HT.sub.2C receptors are coupled to phospholipase C and mediate responses through the phosphatidylinositol pathway. Studies with agonists and antagonists display a wide range of receptor responses suggesting that there is a wide diversity of regulatory mechanisms governing receptor activity. The 5-HT.sub.2A and 5-HT.sub.2C receptors have also been implicated as the site of action of hallucinogenic drugs.
Much of the knowledge about the structure of G protein-coupled receptors has come from the study of the .beta..sub.2 -adrenergic receptor. Over the last several years, site-directed mutagenesis has been used to try to determine the amino acid residues important for ligand binding in both the .beta..sub.2 -adrenergic and 5-HT.sub.2A receptors. In addition, studies have suggested that in a native (inactive) state of G protein-coupled receptors, the third intracellular loop is tucked into the receptor and is not available for interaction with the G protein. A change of receptor conformation (active) results in the availability or exposure of the C-terminal region of the third intracellular loop.
In 1990 Cotecchia et al..sup.1 were studying the G protein specificity determining characteristics of the third intracellular loop by creating chimeric receptors in which the third intracellular loops had been exchanged between the .alpha..sub.1 -adrenergic receptor and the .beta..sub.2 -adrenergic receptor. The specific G protein coupled activation was essentially switched between the two receptors. While attempting to determine which portions of the loop were responsible for the specificity, Cotecchia et al. discovered an unexpected phenomena; namely that the modification in the third intracellular loop of the .alpha..sub.1 -adrenergic receptor of three residues, Arg288, Lys290, and Ala293, created a mutant receptor with two orders of magnitude greater affinity for agonist and which coupled to the second messenger system even in the absence of agonist. These modifications were made in the carboxy end of the third cytoplasmic loop adjacent to the sixth transmembrane helix. The changes responsible for this increase were isolated to either a Ala293.fwdarw.Leu or a Lys290.fwdarw.His mutation. Thus, a constitutively active state of a G protein-coupled neuroreceptor had been created. Subsequently, Kjelsberg et al..sup.2 demonstrated that mutation of the amino acid at position 293 in the .alpha..sub.1B -adrenergic receptor to any other of the 19 amino acids also produced a constitutively active state. Subsequently, mutations in the .beta..sub.2 -adrenergic receptor near the carboxy end of the third cytoplasmic loop have also been shown by Samama et al..sup.3 to constitutively activate this receptor.
When foci resulting from constitutively active .alpha..sub.1B -adrenergic receptors were injected into nude mice, tumor formation occurred. Over the past 5 years, since the discovery that several thyroid adenomas contained mutations of the thyroid stimulating hormone (TSH) receptor, constitutively activated receptors have been found associated with several human disease states. The mutations responsible for these disease states have been found in the transmembrane domains and intracellular loops. For the TSH receptor, mutations at 13 different amino acid positions have been found in the transmembrane domain, the third intracellular loop, and the second and third extracellular loops. Clearly, constitutively activating mutations are not limited to the third intracellular loop and the critical site for constitutive activation varies with each G protein-coupled receptor. The importance of the initial observations was well stated in Cotecchia et al..sup.1 : "Such mutations might not only help to illuminate the biochemical mechanisms involved in receptor-G protein coupling but also provide models for how point mutations might activate potentially oncogenic receptors."
In light of the above referenced discoveries, the importance and utility of discovering other constitutively activated neuronal receptors cannot be understated. However, the hope that other neuronal receptors could be easily and readily mutated to a constitutively active form by mutations in the third cytoplasmic loop was destroyed by the report of Burstein et al..sup.4 in 1995 of a comprehensive mutational approach to the G protein coupled M5 muscarinic acetylcholine receptor. In that approach, Burstein et al. had randomly and comprehensively mutated the C-terminal region of the third intracellular loop of the M5 muscarinic acetylcholine receptor, but no constitutive activating mutations were found.
Definition:
CONSTITUTIVELY ACTIVATED RECEPTOR shall mean a G protein-coupled receptor which: 1) exhibits an increase in basal activity of the second messenger pathway in the absence of agonist above the level of activity observed in the wild type receptor in the absence of agonist; 2) may exhibit an increased affinity and potency for agonists; 3) exhibits an unmodified or decreased affinity for antagonists; and 4) exhibits a decrease in basal activity by inverse agonists.